Ruppia mongolica (Ruppiaceae), a new species from Inner Mongolia (China), based on morphological and genetic data

Abstract Ruppia mongolica Y. Zou & X.W. Xu, a new species from Inner Mongolia, China, is described and illustrated. The phylogenetic position of the new species within the genus was analyzed based on eight chloroplast DNA fragments and an ingroup sampling of all Eurasian species of Ruppia. The results showed that R. mongolica formed a separate branch between R. sinensis and the clade of R. maritima, R. brevipedunculata, R. drepanensis, and R. cirrhosa. Based on molecular and geographical evidence, our study reveals that R. mongolica is closely related to R. sinensis and R. brevipedunculata but differs from the former in the length and shape of the peduncle and seed size, and from the latter in the length of the peduncle, number of carpels per flower, and seed size. In addition, the karyotype analysis revealed that R. mongolica is octoploid, which is first reported within Ruppia, further supporting R. mongolica as a new species.


| Molecular analysis
The fresh and healthy young leaves of five individuals of the new species (one from Urad Front Banner and four from Tumote Right Banner) were used for genomic DNA extraction. Paired-end Illumina libraries with insert sizes of ~300 bp were constructed from the extracted DNA using the NEBNext Ultra DNA Library Prep Kit according to the manufacturer's instructions and sequenced on the NovaSeq 6000 platform. Fastp v.0.20.0 was used for quality control and adapter trimming to obtain high-quality reads (Chen et al., 2018).
Sequences of the eight chloroplast DNA fragments were concatenated using PhyloSuite v.1.1.15 (Zhang et al., 2020). Gaps were treated as missing data. The most suitable model for the combined data was determined using ModelFinder implemented in PhyloSuite.
The maximum likelihood (ML) trees were constructed using IQ-TREE implemented in PhyloSuite. The support of the branches was estimated using 1000 bootstrap replicates. Phylogenetic tree was visualized in iTOL v.6.6 (https://itol.embl.de).

| Morphological characters and chromosome number
The morphological characters of the new species differ from all species previously described in Ruppia. The short peduncle of the new species (1.8-3.8 cm, Figures 1 and 2) makes it quite distinct from other Ruppia species, except for R. maritima and R. brevipedunculata, which have similar peduncle length. However, the number of carpels of the new species (6-8, Figures 1 and 2) is evidently more than the latter two species (2-5 in R. maritima and 4 in R. brevipedunculata).
The new species can be distinguished from two Chinese species, R. sinensis and R. brevipedunculata, in several traits: differing from R. sinensis in the length and shape of peduncle and seed size, and from R. brevipedunculata in the length of peduncle, number of carpels per flower and seed sizes ( Table 1). The karyotype analysis revealed that the chromosome number of R. mongolica is 2n = 8x = 80, while that of R. sinensis is 2n = 4x = 40 ( Figure 3).

| Diagnosis
The new species is most closely related to R. sinensis and R. brevipedunculata but differs from the former in its short and straight peduncles (vs. long and spirally coiled) and 2.2-3.1-mm-long seeds (vs. 1.5-2.5-mm-long), and differs from the latter in 1.8-3.8-mm-long

| Distribution and habitat
To date, Ruppia mongolica has only been found in the two localities in central Inner Mongolia, China ( Figure 5). It grows in shallow stagnant waters with salinity 3-15 ppt.

| DISCUSS ION
The characteristics in the reproductive architecture are consid-  (Mannino et al., 2015), which is similar to that of R. mongolica (1.8-3.8 cm). However, the number of carpels of R. mongolica (6-8) is more than that of R. maritima (2-5). The recently described species R. bicarpa from South Africa has flowers with only two carpels (Ito et al., 2015), and R. mexicana from Mexico (Den Hartog et al., 2016) has spirally coiled long peduncle (5-25 cm) and 2-4 carpels per flower, which are different from those of R. mongolica.
Among four newly published species, both R. mexicana (Den Hartog et al., 2016) and R. sinensis (Figure 3, our data) are tetraploid. Therefore, the octoploid R. mongolica is first reported within Ruppia.
Although the Australian species R. polycarpa, R. megacarpa, and R. tuberosa and the African species R. bicarpa were not included in our phylogenetic analysis, their phylogenetic position was basal and genetically differed from Eurasian species (Ito et al., 2015).
Within Eurasian species, R. mongolica was reconstructed as a branch between R. sinensis and the clade of R. maritima, R. brevipedunculata, R. drepanensis, and R. cirrhosa with good support (Figure 4). Therefore, based on molecular and geographical evidence, R. mongolica is most closely related to two Chinese species R. sinensis and R. brevipedunculata. However, distinct characteristics in reproductive architecture between R. mongolica and these two species (Table 1)  (supporting). Dan Yu: Conceptualization (supporting); funding acquisition (lead); methodology (supporting); validation (supporting); writing -review and editing (supporting).

ACK N OWLED G M ENTS
We are grateful to Dr. Ya Li for the botanical line drawing, and Dr. Wei Du for taking photos and making specimens. This study was supported by the Special Project of Basic Work of Science and Technology, Ministry of Science and Technology, China (No. 2013FY112300).

CO N FLI C T O F I NTE R E S T S TATE M E NT
We declare that the named authors have no conflicts of interest, financial or otherwise.

DATA AVA I L A B I L I T Y S TAT E M E N T
All raw sequencing data obtained from this study have been deposited in the Genome Sequence Archive in National Genomics Data Center under the accession number: CRA009765. The chloroplast DNA sequences analyzed for this study can be found in the GenBank with accession number OP723916.